ABSTRACT This is an application for a 4 year competitive supplement to add a multimodal MRI scan, at 24 months of age, to a newly funded study that aims to replicate and extend our findings predicting autism spectrum disorder (ASD) in high familial risk (HR) infants using MRI scans in the first year of life. HR infants (who have an older affected sibling) have approximately a 12-fold increase in their risk (1/5 vs 1/59) to develop ASD. In HR infants who develop ASD, the first year of life is a period that is relatively free of the defining behavioral characteristics of the disorder, which then consolidate into full diagnosis over the second year of life and beyond. Longitudinal studies involving structural, diffusion, and functional imaging from the Infant Brain Imaging Study (IBIS) Network (https://www.ibis-network.org/) have produced new and fundamental insights into early brain and behavior changes across 6-24 months of age in infants who later meet criteria for ASD. The IBIS Network received funding in April 2019 to replicate and extend findings showing that structural and functional connectivity (fcMRI) MRI scans at 6 and 12 months of age can accurately (positive predictive values greater than 80%) predict ASD diagnosis at 24 months of age. This new study (IBIS-Early Prediction or ?IBIS-EP?) seeks to advance the field toward a clinically actionable, predictive test for ASD in HR infants. The NIMH- approved budget for IBIS-EP, however, does not include support for a 24 month scan of HR infants. Without a 24 month scan, it will not be possible to replicate or extend IBIS? critical findings about brain development [1-8], that are temporally specific and require the 24-month scan for characterization of the precise timing of brain changes. This competitive supplement would allow us to acquire, process, and analyze 24 month multimodal MRI scans for the 250 HR infants participating in the IBIS-EP study (who are already scheduled to return for 24 month diagnostic assessment). This time sensitive opportunity to leverage resources from IBIS-EP, and data from two parallel studies by IBIS co-investigators involving 80 low-risk (LR) infants, would allow us (Aim 1) to attempt to replicate our previous reports of age-specific, structural, diffusion, and functional connectivity MRI brain changes at 6-12 and 24 months; and (Aim 2) to investigate potential mechanisms underlying previously reported MRI findings in a new, prospective cohort, scanned through 24 months. Studies will examine the development of increased cerebrospinal fluid and aberrant white matter microstructure in ASD vs. HR-negative and LR groups, and test ASD outcome group-based differences in brain functional connectivity. The addition of a 24 month scan would, therefore, dramatically increase the impact of the parent project by: 1) identifying specific developmental windows (i.e., time intervals with significant group differences in change rate of brain features that correspond to later clinical outcomes); 2) identifying biomarkers of homogenous subgroups and symptom progression; and 3) identifying therapeutic targets, e.g., brain volume expansion as a marker of over- proliferation of neural progenitor cells, for study in cross-species and cellular models to aid drug development.